The present invention concerns pre-S gene coded hepatitis B immunogens, vaccines and diagnostics. More especially, this invention concerns novel pre-S gene coded peptides and novel carriers, particularly carriers for pre-S gene coded peptides. Even more especially, the present invention relates to synthetic pre-S gene coded peptides covalently linked to lipid vesicle carriers.
There are approximately 600,000 persistent carriers of hepatitis B virus (HBV) in the United States; the estimated total number of carriers in the world is 200 million. A considerable portion of HBV carriers have chronic liver disease. The involvement of HBV in liver cancer has been demonstrated (W. Szmuness, Prog. Med. Virol. 24, 40 (1978) and R.P. Beasley, L.-Y. Hwang, C.-C. Ling, C.-S. Chien, Lancet Nov., 21, 1129 (1981)).
HBV infections thus represent a major public health problem worldwide. Already available vaccines (S. Krugman, in Viral Hepatitis: Laboratory and Clinical Science, F. Deinhardt, J. Deinhardt, Eds., Marcel Dekker, Inc., New York-Basel, 1983, pp. 257-263) produced from the serum of HBV carriers, because of limited resources and production costs involved, do not provide the appropriate means to control and eradicate the disease worldwide. There is hope, however, that this may be accomplished by vaccines based on recombinant DNA technology and/or synthetic peptides.
The biology, structure and immunochemistry of HBV and the genetic organization of its DNA genome have been reviewed (B.S. Blumberg, Science, 197 17, (1977)). The cloning and sequencing of the genome of several hepatitis virus (HBV) isolates led to the elucidation of the genetic structure of the viral DNA (P. Tiollais, P. Charnay, G.N. Vyas, Science, 213, 406, (1981)).
The immunologic markers of HBV infection include the surface antigen (HBsAg), the core antigen (HBcAg), the "e" antigen (HBeAg) and their respective antibodies. Antibodies against HBsAg are protective against HBV infection.
Several antigenic subtypes of HBV and of subviral approximately 22 nm diameter particles (hepatitis B surface antigen; HBsAg) have been recognized (G. Le Bouvier, A. Williams, Am. J. Med. Sci., 270, 165 (1975)). All of these subtypes (for example, ayw, adyw, adw2, adw and adr) share common (group-specific) envelope epitopes, the immune response against which appears sufficient for protection against infection by any of the virus subtypes (W. Szmuness, C.E. Stevens, E.J. Harley, E.A. Zang, H.J. Alter, P.E. Taylor, A. DeVera, G.T.S. Chen, A. Kellner, et al., N. Enql. J. Med., 307, 1481, (1982)).
The physical structure and proposed genetic organization of the HBV genome are described by Tiollais et al, 1981, supra at pp. 408-409. There are two DNA strands, namely the long (L) strand and the short (S) strand. The L strand transcript has four open reading frame regions which are termed (S+pre-S), C, P and X.
The open reading frame region (S+pre-S) corresponds to the envelope (env) gene of HBV DNA and codes for a family of proteins found in the HBV envelope and in virus related particles.
A schematic representation of the potential translation products of the env gene(s) of HBV DNA is as follows: ##STR1##
The numbers in the above schematic refers to amino acids (AA). A translation initiation site at Met 1 exists for the adw2 and adr substypes only. The first amino acid for the other subtypes correspond to position pre-S 12.
Hereinafter, amino acid sequences corresponding to the pre-S region (env 1 to 174) are designated with the prefic "pre-S" and amino acid sequences corresponding to the S region (env 175 to 400) are designated by the prefix "S"In the env gene product representation, the S region spans amino acids 175 to 400 as compared to amino acids 1 to 226 in the "S region only" representation.
In the above schematic, the pre-S region is defined by amino acid sequence positions pre-S 1 to amino acid sequence position pre-S 174. The S region is defined by sequence positions S 1 (amino acid 175 of the open reading frame and adjacent to pre-S 174) to sequence position S 266 (amino acid 400 of the open reading frame). The s-gene product (S-protein) consists of this 226 amino acid sequence.
The epitope(s) essential for eliciting virus-neutralizing antibodies have not yet been unambiguously defined. It has been reported that the group-specificity is represented by a complex of determinants located on each of the two major approximately 22 and approximately 26 kilodalton constituent proteins (P22 and P26) of the virus envelope and of the hepatitis B surface antigen (HBsAg). See J. W.-K. Shih, J. L. Gerin, J. Immunol., 115, 634, (1975); J. W.-K. Shih, P. L. Tan, J. L. Gerin, J. Immunol., 120, 520, (1978); S. Mishiro, M. Imai, K. Takahashi, A. Machida, T. Gotanda, Y. Miyakawa, M. Mayumi, J. Immunol., 124, 1589, (1980); and G. R. Dreesman, R. Chairez, M. Suarez., F. B. Hollinger, R. J. Courtney, J. L. Melnick, J. Virol., 16, 508, (1975).
These proteins have identical amino acid sequences coded for by the S-gene of HBV DNA (Tiollais et al, supra), but the larger protein also carries carbohydrate chains. Peptides corresponding to selected segments of the S-gene product were synthesized and shown to elicit antibodies to HBsAg (anti-HBs). However, immunization of chimpanzees with these peptides resulted in only partial protection against HBV infection (N. Williams, Nature, 306, 427, (1983)).
It has been reported recently that the minor glycoprotein components of HBsAg with M.sub.r of approximately 33 and approximately 36 kilodaltons (P33, P36) are coded for HBV DNA and contain the sequence of P22 (226 amino acids corresponding to the S region) and have 55 additional amino acids at the amino-terminal part which are coded by the pre-S region of the env gene(s) of HBV DNA. See W. Stibbe, W.H. Gerlich, Virology, 123, 436, (1982); M.A. Feitelson, P.L. Marion, W.S. Robinson, Virology, 130, 76, (1983); W. Stibbe, W.H. Gerlich, J. Virol., 46, 626, (1983); and A. Machida, S. Kishimoto, H. Ohnuma, H. Miyamoto, K. Baba, K. Oda, T. Nakamura, Y. Miyakawa, M. Mayumi, Gastroenterology, 85, 268, (1983). Machida et al describe an amino acid sequence composition as a receptor for polymerized serum albumin.
Heretofore, amino acid sequences coded for by the pre-S region of the hepatitis B virus DNA were virtually completely ignored for purposes of producing synthetic vaccines. The hepatitis B vaccine currently in use in the U.S. lacks the pre-S gene coded sequences (and therefore does not elicit antibodies to such sequences) and thus elicits an immune response to the HBV envelope which is incomplete as compared with that occurring during recovery from natural infection.
The generation of antibodies to proteins by immunization with short peptides having the amino acid sequence corresponding to the sequence of preselected protein fragments appears to be a frequent event (Nima, H. L., Houghten, R. A., Walker, L. E., Reisfeld, R. A., Wilson, I. A., Hogle, J. M. and Lerner, R. A., "Generation Of Protein-Reactive Antibodies By Short Peptides Is An Event Of High Frequency: Implications For The Structural Basis Of Immune Recognition"Proceedings of the National Academy of Sciences USA, 80, 4949-4953, (1983)). Nevertheless, the generation of antibodies which recognize the native protein may depend on the appropriate conformation of the synthetic peptide immunogen and on other factors not yet understood. See Pfaff, E., Mussgay, M., Bohm, H. O., Schulz, G. E. and Schaller, H., "Antibodies Against A Preselected Peptide Recognize And Neutralize Foot And Mouth Disease Virus"The EMBO Journal, 7, 869-874, (1982); Neurath, A.R., Kent, S.B.H. and Strick, N., "Specificity Of Antibodies Elicited By A Synthetic Peptide Having A Sequence In Common With A Fragment Of A Virus Protein, The Hepatitis B Surface Antigen," Proceedings Of The National Academy Of Sciences USA, 79, 7871-7875, (1982); Ionescu-Matiu, I., Kennedy, R.C., Sparrow, J.T., Culwell, A.R., Sanchez, Y., Melnick, J.L. and Dreesman, G.R., "Epitopes Associated With A Synthetic Hepatitis B Surface Antigen Peptide"The Journal Of Immunology, 130, 1947-1952, (1983); and Kennedy, R. C., Dreesman, G. R., Sparrow, J. T., Culwell, A. R., Sanchez, Y., Ionescu-Matiu, I., Hollinger, F. B. and Melnick, J. L. (1983); "Inhibition Of A Common Human Anti-Hepatitis B Surface Antigen Idiotype By A Cyclic Synthetic Peptide," Journal of Virology, 46, 653-655, (1983). For this reason, immunization with synthetic peptide analogues of various virus proteins has only rarely resulted in production of virus-neutralizing antisera comparable to those elicited by the viruses (virus proteins) themselves (Pfaff et al., 1982, supra). Thus, the preparation of synthetic immunogens optimally mimicking antigenic determinants on intact viruses remains a challenge.
Replacement of commonly used protein carriers, namely keyhole limpet hemocyanin (KLH), albumin, etc., by synthetic carriers, represents part of such challenge. Although recent reports indicate that free synthetic peptides can be immunogenic, (Dreesman, G. R., Sanchez, Y., Ionescu-Matiu, I., Sparrow, J. T., Six, H. R., Peterson, D. L., Hollinger, F. B. and Melnick, J. L., "Antibody To Hepatitis B Surface Antigen After A Single Inoculation Of Uncoupled Synthetic HBsAg Peptides" Nature, 295, 158-160, (1982), and Schmitz, H. E., Atassi, H., and Atassi, M.Z., "Production Of Monoclonal Antibodies To Surface Regions That Are Non-Immunogenic In A Protein Using Free Synthetic Peptide As Immunogens: Demonstration With Sperm-whale Myoglobin"Immunological Communications, 12, 161-175, (1983)), even in these cases the antibody response was enhanced by linking of the peptides to a protein carrier (Sanchez, Y., Ionescu-Matiu, I., Sparrow, J.T., Melnick, J.L., Dreesman, G.R., "Immunogenicity Of Conjugates And Micelles Of Synthetic Hepatitis B Surface Antigen Peptides"Intervirology, 18, 209-213, (1982)).
Hepatitis B virus has not yet been propagated in vitro and knowledge concerning its reaction and receptors on target cells remains scant. One of the essential functions of virus surface proteins is the recognition of specific receptors on target cell membranes. The specific attachment of viruses to cells is the essential first step in virus entry into cells. The receptor specificity encoded in restricted regions of the virus surface structure may determine the virus host range, tissue tropism and pathogenesis (K. Lonberg-Holm and L. Philipson, eds. Receptors and Recognition, Series B, Volume 8, Virus Receptors: Part 2 - Animal Viruses. (London: Chapman and Hall), pp. 85-211, (1981); B. N. Fields and M. I. Greene, "Genetic and Molecular Mechanisms of Viral Pathogenesis: Implications for Prevention and Treatment"Nature, 300, 19-23, (1982); A. H. Sharpe and B. N. Fields, "Pathogenesis of Viral Infections: Basic Concepts Derived from the Reovirus Model"New Engl. J. Med., 312, 486-497, (1985)), Cellular receptors for distinct viruses have been identified as receptors for discrete physiologically important ligands (D. A. Eppstein, Y. V. Marsh, A. B. Schreiber, S. R. Newman, G. J. Todaro and J. J. Nestor, "Epidermal Growth Factor Receptor Occupancy Inhibits Vaccinia Virus Infection", Nature, 318, 663-665, (1985)). Therefore, the understanding of the detailed features of cell receptors and the corresponding virus binding sites is an important step in explaining virus-cell interactions.
Furthermore, an effective immune response to the virus surface regions involved in cell receptor recognition, or to neighboring regions, is an important component of the host's virus-neutralizing response, (B. Mandel, Virus Neutralization. In Immunochemistry of Viruses: The Basis for Serodiagnosis and Vaccines, M. H. V. Van Regenmortel and A. R. Neurath, eds. (Amsterdam: Elsevier), pp. 53-70, (1985); A. Baltimore, "Picornaviruses are No Longer Black Boxes, Science, 229, 1366-1367, (1985)). Despite the fundamental biological importance of cell receptor recognition domains on viral surface proteins, the domains of only a few viruses have been defined. Notably, the receptor binding sites on influenza viruses (D. C. Wiley, I. A. Wilson and J. J. Skehel, "Structural Identification of the Antibody-Binding Sites of Hong Kong Influenza Haemagglutinin and their Improvement in Antigenic Variation"Nature, 289,373-378, (1981); I. A. Wilson, J. J. Skehel and D. C. Wiley, "Structure of the Haemagglutinin Membrane Glycoprotein of Influenza Virus at 3 A Resolution"Nature, 289,366-373, (1981)) and picornaviruses (J. M. Hogle, M. Chow and D. J. Filman, "Three-Dimensional Structure of Poliovirus at 2.9 A Resolution"Science, 229, 1358-1365, (1985); M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. R. Griffith, H.-J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry and G. Vriend "Structure of a Human Common Cold Virus and Functional Relationship to Other Picornaviruses, Nature, 317, 45-153, (1985)) have been tentatively assigned to restricted regions of virus surface proteins, based on x-ray crystallographic methods and amino acid sequence data.
Hepatitis B virus (HBV) is a major human pathogen implicated in primary hepatocellular carcinoma. The virus is a member of the group of hepadnaviridae (W.S. Robinson, Hepatitis B Virus. In Viral Hepatitis Laboratory and Clinical Science, F. Deinhardt and J. Deinhardt, eds. (New York: Marcel Dekker, Inc.), pp. 57-116, (1983)), the target of which is the liver. Heretofore, the localization of a hepatocyte receptor recognition site in the HBV env protein was not known.
For commonly used protein carriers there is a strong immune response to the carrier, as well as the synthetic peptide. Thus, it would be advantageous to evoke an anti-HBs response with peptides by use of non-protein carriers, which themselves do not evoke an antibody response.
The possible use of several distinct vaccines in prophylaxis would be facilitated by the availability of fully synthetic immunogens.
______________________________________ DEFINITIONS Amino Acid Code Words (as appearing in FIG. 2) ______________________________________ D Asp aspartic acid N Asn asparagine T Thr threonine S Ser serine E Glu glutamic acid Q Gln glutamine P Pro proline G Gly glycine A Ala alanine C Cys cysteine V Val valine M Met methionine I Ile isoleucine L Leu leucine Y Tyr tyrosine F Phe phenylalanine W Trp tryptophane K Lys lysine H His histidine R Arg arginine HBV hepatitis B virus HBsAG hepatitis B surface antigen. DNA deoxyribonucleic acid ______________________________________